Techniques of crop improvement in agriculture involve a search for strains of plants which exhibit new and useful characteristics, or to refine and improve on existing ones. The search has evolved from mere selection of a desirable parent plant, to hybridization between parental strains in which each exhibits desirable characteristics and to crossbreeding between homozygous strains such that identical F.sub.1 progeny will be produced in each subsequent crossbreeding.
The conventional methods of maintaining genetic identity are well known and described in the literature. See, e.g., R. W. Allard "Principle of Plant Breeding", (John Wiley and Sons, Inc., 1960). The maintenance of purebred strains and the repeated crossbreeding to obtain F.sub.1 progeny are time consuming and labor intensive.
Recent advances in molecular biology have dramatically expanded the ability of scientists to manipulate the germplasm of animals and plants. Genes controlling or contributing to specific phenotypes (for example, genes encoding specific polypeptides that provide antibiotic or herbicide resistance) have been identified within certain germplasm, and have been isolated and characterized. Even more important has been the ability to take the genes which have been isolated from one organism and to introduce them into another organism. This process of transformation can be accomplished even where the recipient organism is from a different phylum, genus or species from that which donated the gene.
Attempts have been made to genetically engineer desired traits into plant genomes by introduction of exogenous genes using genetic engineering techniques. These techniques have been successfully applied in monocotyledonous and dicotyledonous species. The uptake of new DNA by recipient plant cells has been accomplished by various means, including Agrobacterium infection, polyethylene glycol (PEG)-mediated DNA uptake, electroporation of protoplasts, and microprojectile bombardment (for a review see, Songstad, et al., 1995, Plant Cell, Tissue and Organ Culture 40:1-15). Maize transformation is often unreliable or occurs at a fairly low frequency. Any development which could ensure the efficiency of maize transformation would be of tremendous value to the field of crop improvement.
The use of regenerable tissue cultures is well known to those of skill in the art. Plant regeneration from maize callus tissue cultures was first demonstrated by C. E. Green and R. L. Philips in 1975 (Green, et al., 1975, Crop Sci. 15:417-421). Transformation of maize callus can be performed in a variety of ways, including, for example, microprojectile bombardment. It is critical that the tissue used for the transformation, especially in the case of microprojectile bombardment, be of the correct morphology. In maize, Type II or friable callus, at the correct stage physiologically and morphologically, will typically transform at a relatively high efficiency. The difficulty arises in the long term maintenance of Type II callus, for only yellow compact callus that is not watery will transform, and the time required to obtain such callus can be from a few months to a year of subculture (Philips, R. L., et al., In: Sprague, et al. (Eds.), 1988, Corn and Corn Improvement, pp. 345-387, Agronomy, Madison, Wis.). Also, different inbred varieties of maize initiate Type II callus at different rates and are more subject to environmental variation affecting the donor plant and culture conditions (Tomes, D. T., 1985, p. 175-203. In S. W. J. Bright and M. G. K. (Eds.) Advances in agricultural biotechnology: Cereal tissue and cell culture. Nijhoff/Junk, Boston). Cryopreservation of callus would allow one to maintain a supply of regenerable embryogenic cultures, but Type II callus has historically been difficult to cryopreserve (Shillito, R. D., et al., 1994, p. 695-704. In Freeling, M., Walbot, V. (Eds.) The Maize Handbook. Spriger-Verlag, New York). Consequently, being able to capture Type II callus in the most amenable stage for transformation will not only vastly improve the efficiency of transformation but also save the researcher invaluable amounts of time.
Various methods have been described for freezing down plant tissue. In the methods currently available in the art, plant callus is pretreated with sugars or polyols, and a mixture of cryoprotectants (dimehtyl sulfate, proline, sugars, polyols) is applied. This is followed by a slow freezing to -40 degree celsius before quenching in liquid nitrogen and rapid thawing (for a review see, Withers, L. A., 1987, Oxford Surveys of Plant Molecular and Cell Biology, 4:221-272). Such methods require an expensive programmable freezer to obtain adequate results. In addition, cryoprotectants which effect the morphology and physiology of the tissue are typically necessary to prevent lesions which form during freezing and thawing and which subsequently result in cell death (McLellan, et al., 1990, Cryo-Letters 11:189-204). In U.S. Pat. No. 5,596,131, a method for cryopreservafion of embryogenic cell cultures is described. Callus of Dacrylis glomerata L. is mixed with a cryoprotectant solution containing glycerol, dimethyl sulfoxide, and proline and then placed in an apparatus which can freeze the callus at a controlled rate. In the published PCT application WO 95/06128, maize embryogenic cells of Type II callus in suspension culture are cryopreserved by adding a cryoprotectant containing dimethyl sulfoxide, polyethylene glycol, proline and glucose to the suspension cultures and then cooling the mixture at a controlled rate at 0.5 degree per minute. Both of these methods introduce cryoprotectants into the cells resulting in undesirable morphological and physiological changes in the callus. In addition, both methods require an expensive programmable freezing apparatus, to control the rate of cooling.
In other, somewhat related methods, somatic embryos have been preserved for use as artificial seeds. In U.S. Pat. No. 4,615,141, Janick and Kitto describe a method of pre-treating embryos with increasing sucrose concentrations or by applying abscisic acid, followed by encapsulation of one or more embryos in a hydrated coating material. In U.S. Pat. No. 4,777,762, Redenbaugh et al. describe a method for producing dessicated analogs of botanic seeds which are created by removing a portion of the water by slow or fast drying so that the plant tissue is no longer saturated with water. In U.S. Pat. No. 5,464,769, Attree and Fowke describe a method of desiccating conifer somatic embryos wherein the embryos are matured, desiccated and then encapsulated. A variety of publications describe preservation of plant cells by encapsulation of plant cells or embryos, preculture in the presence of an osmoticum, and then dessication by placing coated cell in the laminar flow hood or in the presence of silica gel, followed by immersion in liquid nitrogen (Bachiri, et al., 1995, Plant Cell. Tissue and Organ Culture 43: 241-248; Dumet, et al., 1993, Plant Cell Reports 12: 353-355; Sakai, et al., 1991, Plant Science 74: 243-248; Shimonisih, et al., 1991, Japan J. Breed. 41: 347-351; Uragami, et al., 1993, Cryo-Letters 14: 83-90; Dereddre, et al., 1991, Cryo-Letters 12: 125-143; Scottez, et al., 1992, Cryobiology 29: 691-700; Paulet, et al., 1993, Plant Cell Reports 12: 525-529; Niino, et al., 1992, Plant Science 87: 199-206). Maize somatic embryos have been grown on abscisic acid and then subjected to controlled relative humidity dehydration (Compton et al., 1992, In Vitro Cell. Dev. Biol. 28P: 197-201). None of the above methods uses actively growing, transformable plant tissue as described in the present invention. In addition, the present invention does not require encapsulation, the use of a programmable freezer, or cryoprotectants in the freeze mixture.
One of the advantages of the present invention is to allow the scientist to establish a readily transformable callus culture and keep it for long periods. This is especially important when sources of embryos become unavailable or if the scientist finds a line callus that is readily transformable. In addition, the present invention provides a method that allows for preservation of lines that have already been transformed, but for logistical reasons cannot be immediately progressed to callus growth and plant regeneration. Callus lines can now be frozen at any stage of the process from initial generation of a line, maintenance of the pre-transformation or post-transformation, or for sub-culturing. The present invention is simpler and less expensive to perform than earlier methods and can be used on a variety of callus types, unlike other methods .